U.S. patent application number 12/253877 was filed with the patent office on 2009-05-07 for compositions and assays utilizing adp or phosphate for detecting protein modulators.
Invention is credited to Jeffrey T. Finer, Fady Malik, Roman Sakowicz, Christopher Shumate, Kenneth Wood.
Application Number | 20090117534 12/253877 |
Document ID | / |
Family ID | 40295731 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090117534 |
Kind Code |
A1 |
Finer; Jeffrey T. ; et
al. |
May 7, 2009 |
COMPOSITIONS AND ASSAYS UTILIZING ADP OR PHOSPHATE FOR DETECTING
PROTEIN MODULATORS
Abstract
Described herein are methods which identify candidate agents as
binding to a protein or as a modulator of the binding
characteristics or biological activity of a protein. Generally, the
methods involve the use of ADP or phosphate. The assays can be used
in a high throughput system to obviate the cumbersome steps of
using gels or radioactive materials.
Inventors: |
Finer; Jeffrey T.; (Foster
City, CA) ; Malik; Fady; (Burlingame, CA) ;
Sakowicz; Roman; (Foster City, CA) ; Shumate;
Christopher; (San Francisco, CA) ; Wood; Kenneth;
(Foster City, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
40295731 |
Appl. No.: |
12/253877 |
Filed: |
October 17, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12150596 |
Apr 28, 2008 |
|
|
|
12253877 |
|
|
|
|
11655733 |
Jan 18, 2007 |
7378254 |
|
|
12150596 |
|
|
|
|
11270193 |
Nov 8, 2005 |
7202051 |
|
|
11655733 |
|
|
|
|
10856580 |
May 28, 2004 |
|
|
|
11270193 |
|
|
|
|
10106665 |
Mar 25, 2002 |
6743599 |
|
|
10856580 |
|
|
|
|
09724990 |
Nov 28, 2000 |
|
|
|
10106665 |
|
|
|
|
09314464 |
May 18, 1999 |
6410254 |
|
|
09724990 |
|
|
|
|
Current U.S.
Class: |
435/4 |
Current CPC
Class: |
C12Q 1/34 20130101; C12Q
1/485 20130101; G01N 33/573 20130101; C12Q 1/42 20130101 |
Class at
Publication: |
435/4 |
International
Class: |
C12Q 1/25 20060101
C12Q001/25 |
Claims
1. A method of identifying a candidate agent as a modulator of the
function of a target protein, said method comprises: a) adding a
candidate agent to a mixture comprising the target protein that
directly or indirectly produces ADP or phosphate under conditions
that normally allow the production of ADP or phosphate; b)
subjecting the mixture to an enzymatic reaction that uses said ADP
or phosphate as a substrate under conditions that normally allow
the ADP or phosphate to be utilized; and c) determining the level
of activity of the enzymatic reaction wherein a change in said
level between the presence and absence of said candidate agent
indicates that said candidate agent is a modulator of said target
protein function.
2-26. (canceled)
Description
FIELD OF INVENTION
[0001] This invention is related to the use of adenosine
diphosphate (ADP) or phosphate in assays for identifying compounds
which bind to or modulate the binding characteristics or biological
activity of a protein.
BACKGROUND OF THE INVENTION
[0002] Drugs and other compounds intended for use in the diagnosis,
cure, mitigation, treatment or prevention of disease in man or
other animal or for use in the agricultural arena, have made a
significant impact on the practice of modern medicine and on the
agricultural arena. In some cases, such as in the development of
vaccines, drugs have essentially eradicated once untreatable
diseases. In the case of the agriculture, compounds have been
developed which both extend the life and/or volume of produce as
well as kill unwanted plants where desirable. Therefore, the
development of these compounds is of great interest.
[0003] Many useful compounds modulate the physical interaction of
proteins. Traditionally, these protein-protein interactions have
been evaluated using biochemical techniques, including chemical
cross-linking, co-immunoprecipitation, co-fractionation and
co-purification. Recently genetic systems have been invented to
detect protein-protein interactions. The first work was done in
yeast systems, and was termed the "yeast two-hybrid" system. The
basic system requires a protein-protein interaction in order to
turn on transcription of a reporter gene. Subsequent work was done
in mammalian cells. See Fields et al., Nature 340:245 (1989);
Vasavada et al., PNAS USA 88:10686 (1991); Fearon et al., PNAS USA
89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien
et al., PNAS USA 88:9578 (1991); and U.S. Pat. Nos. 5,283,173,
5,667,973, 5,468,614, 5,525,490, and 5,637,463.
[0004] In another approach to drug discovery, studies are designed
to determine the biological activity of a protein. For example, the
conditions such as the specific substrate or stimulator required
for an enzymatic reaction are investigated. Moreover, there are a
number of studies designed specifically for aide in the detection
step in these assays. For example, one study discloses a
spectrophotometric assay for inorganic phosphate (Pi) to probe the
kinetics of Pi release from biological systems such as GTPases and
ATPases. Webb, PNAS, 89:4884-4887 (1992). Another study reports on
an enzymatic assay of inorganic phosphate in serum using nucleoside
phosphorylase and xanthine oxidase. Ungerer, et al., Elsevier
Clinica Chimica Act, 223:149-157 (1993). A continuous
spectrophotometric assay for aspartate transcarbamylase and ATPases
is reported on in Rieger, et al., Anal. Biochem., 246:86-95 (1997).
There is also a study which reports on the measurement of inorganic
phosphate release using fluorescent probes and its application to
actomysin subfragment 1 ATPase. Brune, et al., Biochem.,
33:8262-8271 (1994). U.S. Pat. No. 4,923,796 discloses a method for
quantitative enzymatic determination of ADP. Microtubule-stimulated
adenosine triphosphate (ATP) hydrolysis by kinesin is discussed in
Hackney, J. Biol. Chem., 269(23):16508-16511 (1994). Furthermore,
enzymatic fluorimetry and fluorimetric assays for ATPase activity
are reported on in Greengard, Nature, 178:632-634 (1956) and Utpal
and Siddhrtha, Biochem. J., 266:611-614 (1990), respectively.
[0005] In a different approach, modulators of an enzymatic reaction
are investigated, wherein the conditions which allow the enzymatic
reaction to occur are already known. For example, U.S. Pat. No.
5,759,795 discloses an assay for identifying an inhibitor of a
Hepatitis C Virus NS3 protein ATPase which involves a luciferase
reaction. Luciferase reactions are known in the art. In the case of
an ATPase inhibitor, the presence of an ATPase inhibitor is
indicated when ATP is available to drive the oxidation of luciferon
by luciferase. This approach requires ATP but does not re-generate
ATP.
[0006] Thus, while efforts have been made toward drug discovery,
more efficient means are desirable. In particular, there is a need
for an efficient system which can distinguish between a compound
directly binding to a second component, or whether the compound
modulates the binding between two other components, or whether the
compound modulates the biological activity of a known enzymatic
reaction. Accordingly, it is an object of the present invention to
provide methods of identifying compounds which either bind to or
which modulate the binding characteristics or the biological
activity of a target protein. It is also an object to provide
compositions for use in the assays provided herein.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods which identify
candidate agents that bind to a a protein or act as a modulator of
the binding characteristics or biological activity of a protein. In
one embodiment, the method is performed in plurality
simultaneously. For example, the method can be performed at the
same time on multiple assay mixtures in a multi-well screening
plate as further described below. Furthermore, in a preferred
embodiment, fluorescence or absorbance readouts are utilized to
determine enzymatic activity. Thus, in one aspect, the invention
provides a high throughput screening system.
[0008] In one embodiment, the present invention provides a method
of identifying a candidate agent as a modulator of the activity of
a target protein. The method comprises adding a candidate agent to
a mixture comprising a target protein which directly or indirectly
produces ADP or phosphate under conditions which normally allow the
production of ADP or phosphate. The method further comprises
subjecting the mixture to an enzymatic reaction which uses said ADP
or phosphate as a substrate under conditions which normally allow
the ADP or phosphate to be utilized and determining the level of
activity of the enzymatic reaction. A change in the level between
the presence and absence of the candidate agent indicates a
modulator of the target protein.
[0009] In one aspect, the target protein indirectly produces the
ADP or phosphate by producing a substrate for a reaction which
produces the ADP or phosphate. In another aspect, the target
protein indirectly produces phosphate or ADP or phosphate by
regulating an enzyme which produces ADP or phosphate. In yet a
further aspect, the target protein directly produces phosphate or
ADP.
[0010] In another aspect, the invention provides a method of
identifying a candidate agent as a modulator of the activity of a
target protein wherein the target protein uses ADP or phosphate
directly or indirectly. The method comprises adding a candidate
agent to a mixture comprising the target protein under conditions
which normally allow the utilization of ADP or phosphate. The
method further comprises determining the level of utilization
wherein a change in the level between the presence and absence of
the candidate agent indicates a modulator of the target
protein.
[0011] In another embodiment provided herein is a method for
identifying whether any two target proteins interact. The method
comprises providing a first target chimera comprising a functional
molecular motor binding domain and a first target protein. The
method further comprises providing a second target chimera
comprising a functional microtubule stimulated ATPase domain and a
second target protein. Additionally, the method comprises combining
the first and second target chimeras under conditions which
normally allow activity of a motor protein which comprises a
molecular motor binding domain and a microtubule stimulated ATPase
domain, wherein an increase in motor protein activity indicates
interaction between the two target proteins.
[0012] In a further embodiment a method is provided for identifying
whether a candidate agent is a modulator of at least one of any two
target proteins. The method comprises providing a first target
chimera comprising a functional molecular motor binding domain and
a first target protein and further providing a second target
chimera comprising a functional microtubule stimulated ATPase
domain and a second target protein. Additionally, the method
comprises combining the first and second target chimeras in the
presence and absence of a candidate, wherein a change in motor
protein activity, which requires both a molecular motor binding
domain and a microtubule stimulated ATPase, between the presence
and absence of a candidate agent indicates the candidate agent is a
modulator of at least one of the target proteins.
[0013] Additionally, provided herein is a chimeric protein
comprising a functional molecular motor binding domain and a target
binding domain wherein the chimeric protein is independent of a
functional microtubule stimulated ATPase domain. Also provided
herein is a chimeric protein comprising a functional microtubule
stimulated ATPase domain and a target binding domain, wherein the
chimeric protein is independent of a functional molecular motor
binding domain.
[0014] In one aspect, a nucleic acid comprising a nucleic acid
encoding a chimeric protein in accordance with the present
invention is provided. In another aspect a cell comprising a
nucleic acid or a chimeric protein in accordance with the present
invention is provided.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The present invention provides methods for the
identification of candidate agents that bind to a target protein or
serve as modulators of the biological activity of a target protein.
These assays utilize various methods to measure, in ways amenable
to high throughput screening, the generation or consumption of ADP
or phosphate. That is, target proteins that either directly or
indirectly produce or consume ADP or phosphate may be screened in
the present invention. Thus, by providing assay systems that
rapidly, efficiently and inexpensively assay ADP or phosphate,
modulators (including both antagonists and agonists) of any test
protein that directly or indirectly produces ADP or phosphate may
be found. The present invention thus utilizes high throughput
assays that obviate the traditional cumbersome steps of using gels
or radioactive materials.
[0016] Accordingly, the present invention provides methods of
screening of target proteins. By "target protein" herein is meant a
protein that directly or indirectly produces ADP or phosphate. The
target proteins can be from eukaryotes or procaryotes, including
mammals, fungi, bacteria, insects, and plants, as well as viruses.
In a preferred embodiment, the target proteins are from mammalian
cells, with rodents (mice, rats, hamsters, guinea pigs and gerbils
being preferred), primates and humans being preferred, and humans
being particularly preferred.
[0017] "Protein" in this context means a compound that comprises at
least two covalently attached amino acids and includes proteins,
polypeptides, oligopeptides and peptides. The proteins may be made
up of naturally occurring amino acids and peptide bonds, or
synthetic peptidomimetic structures. Thus "amino acid", or "peptide
residue", as used herein means both naturally occurring and
synthetic amino acids. For example, homo-phenylalanine, citrulline
and noreleucine are considered amino acids for the purposes of the
invention. "Amino acid" also includes imino acid residues such as
proline and hydroxyproline. The side chains may be in either the
(R) or the (S) configuration. In the preferred embodiment, the
amino acids are in the (S) or L-configuration. If non-naturally
occurring side chains are used, non-amino acid substituents may be
used, for example to prevent or retard in vivo degradations.
[0018] Suitable target proteins, include, but are not limited to,
cytoskeletal proteins including, but not limited to, kinesins,
myosins, tubulins, actins, tropomyosins, and troponins, with human
proteins being preferred.
[0019] In a preferred embodiment, the target protein is a kinesin,
including mitotic kinesins. Mitotic kinesins are enzymes essential
for assembly and function of the mitotic spindle, but are not
generally part of other microtubule structures, such as nerve
processes. Mitotic kinesins play essential roles during all phases
of mitosis. These enzymes are "molecular motors" that translate
energy released by hydrolysis of ATP into mechanical force which
drives the directional movement of cellular cargoes along
microtubules. The catalytic domain sufficient for this task is a
compact structure of approximately 340 amino acids. During mitosis,
kinesins organize microtubules into the bipolar structure that is
the mitotic spindle. Kinesins mediate movement of chromosomes along
spindle microtubules, as well as structural changes in the mitotic
spindle associated with specific phases of mitosis. Experimental
perturbation of mitotic kinesin function causes malformation or
dysfunction of the mitotic spindle, frequently resulting in cell
cycle arrest. From both the biological and enzymatic perspectives,
these enzymes are attractive targets for the discovery and
development of novel anti-mitotic chemotherapeutics.
[0020] Suitable kinesins include, but are not limited to, Kin2,
chromokinesin, Kif1A, KSP, CENP-E, MCAK, HSET and Kif15 is
provided. K335, Q475, D679, FL1, P166, H195, FL2, E433, R494, E658,
L360, K491, S553, M329, T340, S405, V465, T488, M1, M2, M3, M4, M5,
M6, FL3, A2N370, A2M511, K519, E152.2, Q151.2, Q353, M472 and
MKLP1. It is understood that unless a particular species is named,
the term "kinesin" includes homologs thereof which may have
different nomenclature among species. For example, the human
homolog of Kif1A is termed ATSV, the human homologue of Xenopus Eg5
is termed KSP, and human HSET corresponds to Chinese hamster
CHO2.
[0021] By "kinesin protein activity" or grammatical equivalents
herein is meant one of kinesin protein's biological activities,
including, but not limited to, its ability to affect ATP
hydrolyzation. Other activities include microtubule binding,
gliding, polymerization/depolymerization (effects on microtubule
dynamics), binding to other proteins of the spindle, binding to
proteins involved in cell-cycle control, or serving as a substrate
to other enzymes, such as kinases or proteases and specific kinesin
cellular activities such as chromosome congregation, axonal
transport, etc.
[0022] Methods of performing motility assays are well known to
those of skill in the art (see, e.g., Hall, et al. (1996), Biophys.
J., 71: 3467-3476, Turner et al., 1996, Anal. Biochem. 242
(1):20-5; Gittes et al., 1996, Biophys. J. 70(1): 418-29; Shirakawa
et al., 1995, J. Exp. Biol. 198: 1809-15; Winkelmann et al., 1995,
Biophys. J. 68: 2444-53; Winkelmann et al., 1995, Biophys. J. 68:
72S, and the like).
[0023] In a preferred embodiment, the target protein directly or
indirectly produces ADP and/or phosphate. Included in the
definition of adenosine diphosphate (ADP) are ADP analogs,
including, but not limited to, deoxyadenosine diphosphate (dADP)
and adenosine analogs. As used herein, phosphate is used
interchangeably with inorganic phosphate.
[0024] In a preferred embodiment, the target protein directly
produces ADP or phosphate. In a preferred embodiment, the target
protein is an enzyme having activity which produces ADP and/or
phosphate as a reaction product. For example, proteins which
directly produce ADP include but are not limited to ATPases,
kinases, GTPases, phosphatases and phosphorylases. Suitable ATPases
include, but are not limited to, myosins, kinesins, dyneins, DNA
gyrase, DNA helicase, topoisomerase I and II, Na+-K+ ATPase, Ca2+
ATPase, F1 subunit of ATP synthase, terminase/DNA packaging
protein; recA, heat shock proteins, NSF, katanin, SecA,
5-lipoxygenase, and actin. Suitable kinases include, but are not
limited to, tyrosine kinases; serine-threonine kinases; receptor
tyrosine kinases; growth factor receptors including but not limited
to insulin receptor, epidermal growth factor receptor, platelet
derived growth factor receptor and fibroblast growth factor
receptor; ErbB2; calmodulin dependent protein kinases; protein
kinase A; protein kinase C; myosin light chain kinase; CDK2 kinase;
ROCK1 kinases; Src kinases; phosphorylase kinase; CheA; adenylate
kinase; glycolytic kinases; EIF-2 alpha protein kinases; and Abl.
Suitable GTPases include, but are not limited to, G proteins, Rho
family GTPases: cdc42, RalA, RhoA and Rac1; Ras proteins;
elongation factors including EF1.alpha., EF1.beta..gamma., EF-Tu
and EF-G; septins; tubulin; ARF related GTPase; rab; SSRP receptor;
rhodopsin; transducin; and GTPase activating protein (GAP).
Suitable phosphatases include, but are not limited to, protein
phosphatases; myosin phosphatase; IP3 phosphatase; pyrophosphatase;
and Cdc25. Suitable phosphorylases include, but are not limited to,
polynucleotide phosphorylase and glycogen phosphorylase.
[0025] By "ATPase" herein is meant an enzyme that hydrolyzes ATP.
For example, ATPases include proteins comprising molecular motors
such as kinesins, myosins and dyneins. "Molecular motor" is a
molecule that utilizes chemical energy to produce mechanical force
or movement; molecular motors are particularly of interest in
cytoskeletal systems. For further review, see, Vale and Kreis,
1993, GUIDEBOOK TO THE CYTOSKELETAL AND MOTOR PROTEINS New York:
Oxford University Press; Goldstein, 1993, Ann. Rev. Genetics 27:
319-351; Mooseker and Cheney, 1995, Annu. Rev. Cell Biol. 11:
633-675; Burridge et al., 1996, Ann. Rev. Cell Dev. Biol. 12:
463-519.
[0026] In one embodiment, the target protein indirectly produces
ADP or phosphate. In one aspect, a target protein indirectly
produces ADP or phosphate by producing a product that then serves
as a substrate in a subsequent enzymatic reaction for producing ADP
or phosphate. For example, in a preferred embodiment, the target
protein can be a pyrophosphate producing enzyme. Suitable
pyrophosphate producing enzymes include, but are not limited to,
DNA polymerases; RNA polymerases; reverse transcriptase; DNA
ligase; adenylate cyclase; guanylate cyclase; PRPP synthetase; TRNA
synthetases; acyl CoA synthetase and acetyl CoA carboxylase.
Similarly, some ATPases produce AMP that can then be used to make
ADP.
[0027] In another embodiment, the target protein is a synthase.
Thus, preferred substrates for producing phosphate include
pyrophosphate and any of the mono-, di- and triphosphate versions
of CTP, UTP, GTP, ATP, and TTP, as well as derivatives including
dideoxy derivatives. Additionally, other sources of substrates that
can be cleaved to phosphate include phosphorylated peptides,
oligonucleotides, carbohydrates, lipids, etc. For example, inositol
triphosphate (IP3) is an important signaling moiety. Accordingly,
any target protein which produces these compounds or others that
can be used to produce phosphate or ADP may be assayed using the
methods of the present invention.
[0028] In another aspect, a target protein indirectly produces ADP
or phosphate by regulating an enzyme which produces phosphate or
ADP. For example, the target can be an activator of an ATPase, such
as an actin filament or a microtubule; thus in this embodiment, the
target protein may be a protein polymer or oligomer. Alternatively,
the target protein can be a filament binding protein or regulatory
protein. For example, the regulatory protein can be the
troponin-tropomyosin complex which regulates the binding of myosin
to actin. Since myosin's ATPase is activated by binding to actin,
modulators of this regulatory protein complex can be identified by
the methods provided herein.
[0029] In a preferred embodiment, the target protein may consume
ADP or phosphate; that is, rather than looking for an increase in
signal, a loss of signal may be monitored.
[0030] Also included within the definition of the target proteins
of the present invention are amino acid sequence variants of
wild-type target proteins. These variants fall into one or more of
three classes: substitutional, insertional or deletional variants.
As for the target proteins as discussed below, these variants
ordinarily are prepared by site specific mutagenesis of nucleotides
in the DNA encoding the target protein, using cassette or PCR
mutagenesis or other techniques well known in the art, to produce
DNA encoding the variant, and thereafter expressing the DNA in
recombinant cell culture as outlined above. However, variant target
protein fragments having up to about 100-150 residues may be
prepared by in vitro synthesis using established techniques. Amino
acid sequence variants are characterized by the predetermined
nature of the variation, a feature that sets them apart from
naturally occurring allelic or interspecies variation of the target
protein amino acid sequence. The variants typically exhibit the
same qualitative biological activity as the naturally occurring
analogue, although variants can also be selected which have
modified characteristics as will be more fully outlined below.
[0031] While the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed variants screened
for the optimal combination of desired activity. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example, M13 primer
mutagenesis and PCR mutagenesis. Screening of the mutants is done
using assays of target protein activities.
[0032] Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about 1 to 20 amino
acids, although considerably larger insertions may be tolerated.
Deletions range from about 1 to about 20 residues, although in some
cases deletions may be much larger.
[0033] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative. Generally
these changes are done on a few amino acids to minimize the
alteration of the molecule. However, larger changes may be
tolerated in certain circumstances. When small alterations in the
characteristics of the target protein are desired, substitutions
are generally made in accordance with the following chart:
TABLE-US-00001 CHART I Original Residue Exemplary Substitutions Ala
Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro
His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu,
Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile,
Leu
[0034] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those shown in Chart I. For example, substitutions may be made
which more significantly affect: the structure of the polypeptide
backbone in the area of the alteration, for example the
alpha-helical or beta-sheet structure; the charge or hydrophobicity
of the molecule at the target site; or the bulk of the side chain.
The substitutions which in general are expected to produce the
greatest changes in the polypeptide's properties are those in which
(a) a hydrophilic residue, e.g. seryl or threonyl, is substituted
for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g. lysyl, arginyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g. glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.
phenylalanine, is substituted for (or by) one not having a side
chain, e.g. glycine.
[0035] The variants typically exhibit the same qualitative
biological activity, although variants also are selected to modify
the characteristics of the target proteins as needed.
Alternatively, the variant may be designed such that the biological
activity of the target protein is altered. For example,
glycosylation sites may be altered or removed.
[0036] Further included within the definition of the target
proteins of the invention are covalent modifications of the target
proteins. One type of covalent modification includes reacting
targeted amino acid residues of a target protein with an organic
derivatizing agent that is capable of reacting with selected side
chains or the N- or C-terminal residues of a target protein.
Derivatization with bifunctional agents is useful, for instance,
for crosslinking the target protein to a water-insoluble support
matrix or surface. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxy-succinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0037] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0038] Another type of covalent modification of the target proteins
included within the scope of this invention comprises altering the
native glycosylation pattern of the polypeptide. "Altering the
native glycosylation pattern" is intended for purposes herein to
mean deleting one or more carbohydrate moieties found in the target
native sequence, and/or adding one or more glycosylation sites that
are not present in the native sequence.
[0039] Addition of glycosylation sites to target polypeptides may
be accomplished by altering the amino acid sequence thereof. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequence (for O-linked glycosylation sites). The target
amino acid sequence may optionally be altered through changes at
the DNA level, particularly by mutating the DNA encoding the target
polypeptide at preselected bases such that codons are generated
that will translate into the desired amino acids.
[0040] Another means of increasing the number of carbohydrate
moieties on the target polypeptide is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0041] Removal of carbohydrate moieties present on the target
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by
Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exo-glycosidases as described by Thotakura
et al., Meth. Enzymol., 138:350 (1987).
[0042] Another type of covalent modification of target proteins
comprises linking the target polypeptide to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0043] Target polypeptides of the present invention may also be
modified in a way to form chimeric molecules comprising a target
protein fused to another, heterologous polypeptide or amino acid
sequence, a preferred embodiment of which is described more fully
below. In one embodiment, such a chimeric molecule comprises a
fusion of a target polypeptide with a tag polypeptide which
provides an epitope to which an anti-tag antibody can selectively
bind. The epitope tag is generally placed at the amino- or
carboxyl-terminus of the target polypeptide. The presence of such
epitope-tagged forms of a target polypeptide can be detected using
an antibody against the tag polypeptide. Also, provision of the
epitope tag enables the target polypeptide to be readily purified
by affinity purification using an anti-tag antibody or another type
of affinity matrix that binds to the epitope tag.
[0044] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et al., BioTechnology, 16:1204-1210 (1988)];
the KT3 epitope peptide [Martin et al., Science, 255:192-194
(1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem.,
266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag
[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397
(1990)].
[0045] As will be appreciated by those in the art, the target
proteins can be made in a variety of ways, including both synthesis
de novo and by expressing a nucleic acid encoding the target
protein.
[0046] Numerous suitable methods for recombinant protein
expression, including generation of expression vectors, generation
of fusion proteins, introducing expression vectors into host cells,
protein expression in host cells, and purification methods are
known to those in the art and are described, for example, in the
following textbooks: Sambrook et al., Molecular Cloning: A
Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,
1989), Ausubel et al., Short Protocols in Molecular Biology (John
Wiley & Sons, Inc., 1995), Harlow and Lane, Antibodies: A
Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,
1988), O'Reilly et al., Baculovirus Expression Vectors: A
Laboratory Manual (New York: Oxford University Press, 1994),
Richardson, Baculovirus Expression Protocols (Totowa: Humana Press,
1995), Kriegler, Gene Transfer and Expression: A Laboratory Manual
(New York: Oxford University Press, 1991), Roth, Protein Expression
in Animal Cells, Methods in Cell Biology Vol. 43 (San Diego:
Academic Press, 1994), Murray, Gene Transfer and Expression
Protocols, Methods in Molecular Biology, Vol. 7 (Clifton: Humana
Press, 1991), Deutscher, Guide to Protein Purification, Methods in
Enzymology Vol. 182 (San Diego: Academic Press, Inc., 1990), Harris
and Angal, Protein Purification Methods: A Practical Approach
(Oxford: IRL Press at Oxford University Press, 1994), Harris and
Angal, Protein Purification Applications: A Practical Approach
(Oxford: IRL Press at Oxford University Press, 1990), Rees et al.,
Protein Engineering: A Practical Approach (Oxford: IRL Press at
Oxford University Press, 1992) and White, PCR Protocols, Methods in
Molecular Biology, Vol. 15 (Totowa, Humana Press, 1993).
[0047] The selection of host cell types for the expression of
target proteins will depend on the target protein, with both
eukaryotic and procaryotic cells finding use in the invention.
Appropriate host cells include yeast, bacteria, archebacteria,
fungi, plant, insect and animal cells, including mammalian cells.
Of particular interest are Drosophila melangaster cells,
Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus
subtilis, SF9 cells (and other related cells for use with
baculoviral expression systems), C129 cells, 293 cells, Neurospora,
BHK, CHO, COS, Dictyostelium, etc.
[0048] In a preferred embodiment, the target proteins are purified
for use in the assays, as outlined herein, to provide substantially
pure samples. By "substantially pure" or "isolated" herein is meant
that the protein is unaccompanied by at least some of the material
with which it is normally associated in its natural state,
preferably constituting at least about 0.5%, more preferably at
least about 5% by weight of the total protein in a given sample. A
substantially pure protein comprises at least about 75% by weight
of the total protein, with at least about 80% being preferred, and
at least about 90% being particularly preferred. Alternatively, the
target protein need not be substantially pure as long as the sample
comprising the target protein is substantially free of other
components that can contribute to the production of ADP or
phosphate (or, in the case of indirect assays, other components
which are subsequently assayed).
[0049] The target proteins may be isolated or purified in a variety
of ways known to those skilled in the art depending on what other
components are present in the sample. Standard purification methods
include electrophoretic, molecular, immunological and
chromatographic techniques, including ion exchange, hydrophobic,
affinity, and reverse-phase HPLC chromatography, and
chromatofocusing. For example, the target protein may be purified
using a standard anti-target antibody column. Ultrafiltration and
diafiltration techniques, in conjunction with protein
concentration, are also useful. For general guidance in suitable
purification techniques, see Scopes, R., Protein Purification,
Springer-Verlag, NY (1982).
[0050] Suitable purification schemes for some specific kinesins are
outlined in U.S. Ser. No. 09/295,612, filed Apr. 20, 1999, hereby
expressly incorporated herein in its entirety, along with
referenced materials.
[0051] The present invention provides methods for screening for
modulators of target proteins. By "modulators" herein is meant both
antagonists and agonists of the target protein. Thus, "modulating
the activity of the target protein" includes an increase in target
protein activity, a decrease in target protein activity, or a
change in the type or kind of activity present. Generally, the
modulator will both bind to the target protein (although this may
not be necessary), and alter its biological or biochemical activity
as defined herein. For inhibitors, changes of 25%, 50%, 75% and
most preferably 100% of at least one biological activity of the
target protein is seen. For activators, preferably the change is a
change of at least 40%, more preferably at least 60%, more
preferably at least 80%, more preferably at least 100%, more
preferably at least 200%, and most preferably by at least 500%.
[0052] Accordingly, the present invention provides methods for
screening candidate bioactive agents for the ability to modulate a
target protein's activity. By "candidate agent" or "candidate
bioactive agent" or "drug candidate" or grammatical equivalents
herein is meant any molecule, e.g., protein, oligopeptide, small
organic molecule, polysaccharide, polynucleotide to be tested in a
screening assay.
[0053] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines (including derivatives, structural
analogs, or combinations thereof), derivatives, structural analogs
or combinations thereof.
[0054] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0055] In an embodiment provided herein, the candidate bioactive
agents are proteins. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. For example, homo-phenylalanine,
citrulline and noreleucine are considered amino acids for the
purposes of the invention. "Amino acid" also includes imino acid
residues such as proline and hydroxyproline. The side chains may be
in either the (R) or the (S) configuration. In the preferred
embodiment, the amino acids are in the (S) or L-configuration. If
non-naturally occurring side chains are used, non-amino acid
substituents may be used, for example to prevent or retard in vivo
degradations.
[0056] In another embodiment, the candidate bioactive agents are
naturally occurring proteins or fragments of naturally occurring
proteins. Thus, for example, cellular extracts containing proteins,
or random or directed digests of proteinaceous cellular extracts,
may be used. In one embodiment, the libraries are of bacterial,
fungal, viral, and mammalian proteins, with the latter being
preferred, and human proteins being especially preferred.
[0057] In one embodiment, the candidate agents are peptides of from
about 2 to about 30 amino acids, with from about 5 to about 20
amino acids being preferred, and from about 7 to about 15 being
particularly preferred. The peptides may be digests of naturally
occurring proteins as is outlined above, random peptides, or random
peptides. By randomized or grammatical equivalents herein is meant
that each nucleic acid and peptide consists of essentially random
nucleotides and amino acids, respectively. Since generally these
random peptides (or nucleic acids, discussed below) are chemically
synthesized, they may incorporate any nucleotide or amino acid at
any position. The synthetic process can be designed to generate
randomized proteins or nucleic acids, to allow the formation of all
or most of the possible combinations over the length of the
sequence, thus forming a library of randomized candidate bioactive
proteinaceous agents.
[0058] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
[0059] In another embodiment, the candidate agents are nucleic
acids. By nucleic acid or "oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked
together. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141
91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437
(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et
al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid
backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895
(1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen,
Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all
of which are incorporated by reference). Other analog nucleic acids
include those with positive backbones (Denpcy et al., Proc. Natl.
Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.
5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);
Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et
al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, Carbohydrate Modifications in Antisense
Research, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996))
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, Carbohydrate Modifications in Antisense Research, Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments.
[0060] In addition, mixtures of naturally occurring nucleic acids
and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. The nucleic acids may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the
nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xanthine,
hypoxanthine, isocytosine, isoguanine, etc.
[0061] As described above generally for proteins, nucleic acid
candidate agents may be naturally occurring nucleic acids, random
nucleic acids, or biased random nucleic acids. For example, digests
of procaryotic or eukaryotic genomes may be used as is outlined
above for proteins.
[0062] In a preferred embodiment, the candidate bioactive agents
are organic chemical moieties, a wide variety of which are
available in the literature.
[0063] In a preferred embodiment, the candidate agent is a small
molecule. The small molecule is preferably 4 kilodaltons (kd) or
less. In another embodiment, the compound is less than 3 kd, 2 kd
or 1 kd. In another embodiment the compound is less than 800
daltons (D), 500 D, 300 D or 200 D. Alternatively, the small
molecule is about 75 D to 100 D, or alternatively, 100 D to about
200 D.
[0064] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 NWS, Advanced Chem
Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.).
[0065] The present invention provides methods of screening
candidate bioactive agents for 6 modulators of target protein
activity. In a preferred embodiment, the methods are in vitro
methods, utilizing purified or partially purified target proteins.
Alternatively, the methods are in vivo methods, utilizing cells
comprising target nucleic acids that can be expressed to produce
target proteins, particularly when the target protein is either
secreted or on the surface.
[0066] In a preferred embodiment, the methods comprise combining a
target protein and a candidate bioactive agent, and evaluating the
effect on the target protein's activity. By "target protein
activity" or grammatical equivalents herein is meant the biological
activity of the target protein. As will be appreciated by those in
the art, the activity of the target protein will vary with the
target protein chosen, and will be generally ascertainable by one
of skill in the art of the target protein.
[0067] In a preferred embodiment, the methods of the invention
comprise the addition of candidate agents to the target proteins.
In general, this is done under conditions which normally allow the
direct or indirect production of ADP or phosphate by the target
protein. The phrase "under conditions which normally allow
production or utilization of ADP or phosphate" as used herein means
that all of the compositions and conditions are provided to allow
the production or utilization of ADP or phosphate. Thus, the
reaction which directly or indirectly produces or uses ADP or
phosphate would normally occur in the absence of the modulator.
[0068] As will be appreciated by those in the art, the components
are added in buffers and reagents to assay target protein activity
and give optimal signals (i.e. the largest ADP or phosphate signals
possible). Since the methods outlined herein allow kinetic
measurements, the incubation periods are optimized to give adequate
detection signals over the background.
[0069] A "modulator of a target protein which directly or
indirectly produces or uses ADP or phosphate" can be any compound
as described herein in the context of candidate agents which
modulates the target protein's direct or indirect production or use
of ADP or phosphate relative to a control.
[0070] In one aspect, the method comprises subjecting the mixture
to an enzymatic reaction which uses ADP or phosphate as a substrate
under conditions which normally allow the ADP or phosphate to be
utilized and determining the level of activity of the enzymatic
reaction. This step can be performed in conjunction with
identifying a modulator of a target protein which directly or
indirectly produces ADP or phosphate or independently thereof to
identify a modulator of a protein which uses ADP or phosphate.
[0071] The phrase to "use ADP or phosphate" as used herein means
that the ADP or phosphate are directly acted upon. In one case, the
ADP, for example, can be hydrolyzed or can be phosphorylated. As
another example, the phosphate can be added to another compound. As
used herein, in each of these cases, ADP or phosphate is acting as
a substrate.
[0072] There are a number of enzymatic reactions known in the art
which use ADP as a substrate. For example, kinase reactions such as
pyruvate kinases are well known. Nature, 78:632 (1956); Mol.
Pharmacol, 6(1):31-40 (1970). This is a preferred method in that it
allows the regeneration of ATP. In one embodiment, the level of
activity of the enzymatic reaction is determined directly. For
example, in a pyruvate kinase reaction, pyruvate or ATP can be
measured by conventional methods known in the art.
[0073] In a preferred embodiment, the level of activity of the
enzymatic reaction which uses ADP as a substrate is measured
indirectly by being coupled to another reaction. For example, in
one embodiment, the method further comprises a lactate
dehydrogenase reaction under conditions which normally allow the
oxidation of NADH, wherein said lactate dehydrogenase reaction is
dependent on the pyruvate kinase reaction. Measurement of enzymatic
reactions by coupling is known in the art, i.e., Nature, 178:632
(1956) and is further discussed below in regards to
fluorescence.
[0074] Furthermore, there are a number of reactions which utilize
phosphate. Examples of such reactions include a purine nucleoside
phosphorylase reaction. This reaction can be measured directly or
indirectly. The reaction can be measured directly by conventional
methods known in the art.
[0075] In a preferred embodiment, the level of activity of the
enzymatic reaction which uses phosphate as a substrate is measured
indirectly by being coupled to another reaction. For example, in
one embodiment, the method further comprises a purine analog
cleavage reaction under conditions which normally allow the
cleavage of the purine analog. See, PNAS, 89:4884-4887 (1992);
Anal. Biochem., 246:86-95 (1997); Biochem., J., 266:611-614 (1990).
Alternatively, xanthine oxidase can be used in conjunction with
purine nucleoside phosphorylase to couple phosphate production to a
change in the absorbance of a substrate for xanthine oxidase. Clin.
Chim. Acta., 223:149-157 (1993).
[0076] In one embodiment, the detection of the ADP or phosphate
proceeds non-enzymatically, for example by binding or reacting the
ADP or phosphate with a detectable compound. For example,
phosphomolybdate based assays may be used which involve conversion
of free phosphate to a phosphomolybdate complex. J. Biol. Chem.,
66:375-400 (1925). One method of quantifying the phosphomolybdate
is with malchite green. Chin. Chim. Acta, 14:361-366 (1966).
Alternatively, a fluorescently labeled form of a phosphate binding
protein, such as the E. coli phosphate binding protein, can be used
to measure phosphate by a shift in its fluorescence.
[0077] In a preferred embodiment, detection of the assay is done
using a detectable label. By "labeled" herein is meant that a
compound has at least one element, isotope or chemical compound
attached to enable the detection of the compound. In general,
labels fall into three classes: a) isotopic labels, which may be
radioactive or heavy isotopes; b) magnetic, electrical, thermal;
and c) colored or luminescent dyes; although labels include enzymes
and particles such as magnetic particles as well. The dyes may be
chromophores or phosphors but are preferably fluorescent dyes,
which due to their strong signals provide a good signal-to-noise
ratio for detection. Suitable dyes for use in the invention
include, but are not limited to, fluorescent lanthanide complexes,
including those of Europium and Terbium, fluorescein, rhodamine,
tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,
Cascade Blue.TM., Texas Red, and derivatives thereof, and others
described in the 6th Edition of the Molecular Probes Handbook by
Richard P. Haugland, hereby expressly incorporated by
reference.
[0078] The invention provides methods of screening candidate agents
for the ability to serve as modulators of target protein activity.
In a preferred embodiment, high throughput screening (HTS) systems
are used, which can include the use of robotic systems. The assays
of the present invention offer the advantage that many samples can
be processed in a short period of time. For example, plates having
96 or as many wells as are commercially available can be used.
[0079] High throughput screening systems are commercially available
(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical
Industries, Mentor, Ohio; Beckman Instruments, Inc., Fullerton,
Calif.; Precision Systems, Inc., Natick, Mass., etc.) These systems
typically automate entire procedures including all sample and
reagent pipetting, liquid dispensing, timed incubations, and final
readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high throughput and rapid
start up as well as a high degree of flexibility and customization.
The manufacturers of such systems, i.e., Zymark Corp., provide
detailed protocols for the various high throughput assays.
[0080] Generally a plurality of assay mixtures are run in parallel
with different agent concentrations to obtain a differential
response to the various concentrations. Typically, one of these
concentrations serves as a negative control, i.e., at zero
concentration or below the level of detection. However, in one
embodiment, any concentration can be used as the control for
comparative purposes.
[0081] In one preferred embodiment, high throughput screening
methods involve providing a library containing a large number of
compounds (candidate compounds) potentially having the desired
activity. Such "combinatorial chemical libraries" are then screened
in one or more assays, as described herein, to identify those
library members (particular chemical species or subclasses) that
display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics or
agricultural compounds.
[0082] For example, in one embodiment, candidate agents are assayed
in highly parallel fashion by using multiwell plates by placing the
candidate agents either individually in wells or testing them in
mixtures. Assay components, such as for example, molecular motors,
protein filaments, coupling enzymes and substrates, and ATP can
then be added to the wells and the absorbance or fluorescence of
each well of the plate can be measured by a plate reader. A
candidate agent which modulates the function of the molecular motor
is identified by an increase or decrease in the rate of ATP
hydroylsis compared to a control assay in the absence of that
candidate agent.
[0083] A preferred HTS system is as follows. The system comprises a
microplate input function which has a storage capacity matching a
logical "batch" size determined by reagent consumption rates. The
input device stores and, delivers on command, barcoded assay plates
containing pre-dispensed samples, to a barcode reader positioned
for convenient and rapid recording of the identifying barcode. The
plates are stored in a sequential nested stack for maximizing
storage density and capacity. The input device can be adjusted by
computer control for varying plate dimensions. Following plate
barcode reading, the input device can be adjusted by computer
control for varying plate dimensions. Following plate barcode
reading, the input device transports the plate into the pipetting
device which contains the necessary reagents for the assay.
Reagents are delivered to the assay plate with the pipetting
device. Tip washing in between different reagents is performed to
prevent carryover. A time dependent mixing procedure is performed
after each reagent to effect a homogeneous solution of sample and
reagents. The sequential addition of the reagents is delayed by an
appropriate time to maximize reaction kinetics and readout levels.
Immediately following the last reagent addition, a robotic
manipulator transfers the assay plate into an optical interrogation
device which records one or a series of measurements to yield a
result which can be correlated to an activity associated with the
assay. The timing of the robotic transfer is optimized by
minimizing the delay between "last reagent" delivery and transfer
to the optical interrogation device. Following the optical
interrogation, the robotic manipulator removes the finished assay
plates to a waste area and proceeds to transfer the next plate from
pipetting device to optical interrogation device. Overlapping
procedures of the input device, pipetting device and optical
interrogation device are used to maximize throughput.
[0084] It is understood that the methods provided herein can be
applied to a varied array of target proteins and are not limited to
cytoskeletal component systems. However, for illustrative purposes,
another example of the present invention is to assay for modulators
of the polymerized state of cytoskeletal filament proteins actin or
tubulin. In this example, the candidate agent or mixture comprising
at least one candidate agent is incubated with the filament protein
under conditions that would normally promote either polymerization
or depolymerization. A molecular motor that is activated by the
filament is then added to the assay mixture and its activity is
monitored by ADP or phosphate release as discussed above. Candidate
agents which increase the fraction of the filament protein in a
polymerized state will be identified by an increase in the motor
ATPase and those which increase the fraction of the filament
protein in a depolymerized state will be identified by a decrease
in the motor ATPase.
[0085] It is understood that once a modulator or binding agent is
identified that it can be subjected to further assays to further
confirm its activity. In particular, the identified agents can be
entered into a computer system as lead compounds and compared to
others which may have the same activity. The agents may also be
subjected to in vitro and preferably in vivo assays to confirm
their use in medicine as a therapeutic or diagnostic or in the
agricultural arena.
[0086] In a preferred embodiment, approximately 1000 assays are
performed per hour with very low false negative and false positive
rates, with up to 10,000 assays an hour being preferred and more
than 10,000 assays per hour being particularly preferred. In a
particularly preferred embodiment, at least one or more of the
steps regarding automated liquid handling or preferred assay design
as described herein are included.
[0087] In one embodiment, the method comprises automated liquid
handling.
[0088] In preferred embodiment, an antifoam or a surfactant is
included in the assay mixture and wash solution. Suitable antifoams
include, but are not limited to, antifoam 289 (Sigma), and others
commercially available. Suitable surfactants include, but are not
limited to, Tween, Tritons including Triton X-100, saponins, and
polyoxyethylene ethers. This eliminates bubbles which often result
in conventional methods requiring pipetting into low volume assay
wells. Thus, in a preferred embodiment, the invention includes the
use of an antifoam, detergent or surfactant as a reagent in a high
throughput screens, including, but not limited to the screens of
the invention. Generally the antifoams, detergents or surfactants
are added at a range from about 0.01 ppm to about 10 ppm, with from
about 1 to about 2 ppm being preferred. In a further preferred
embodiment, the invention includes the use of an antifoam,
surfactant or detergent when the assay requires mixing,
particularly physical mixing such as shaking the microtiter plates.
In an additional preferred embodiment, the invention includes the
use of an antifoam, surfactant or detergent when the assay is done
in microtiter plates, particularly plates with 96 wells or more,
including 96, 384 and 1536 plates.
[0089] In another aspect, a round sample well is used. This helps
increase the pathlength for absorbance measurements for a given
assay volume and helps flatten the meniscus of the solution in each
assay well. Preferably, the method comprises vigorous shaking of
the sample plate following the addition of each reagent.
[0090] In a preferred embodiment herein, a preferred assay design
is provided. In one aspect, the preferred assay preferably uses a
multi-time-point (kinetic) assay, with at least two data points
being preferred. As will be appreciated by those in the art, the
interval can be adjusted to correlate with the biological activity
of the protein. In the case of multiple measurements the absolute
rate of the protein activity can be determined, and such
measurements have higher specificity particularly in the presence
of candidate agents which have similar absorbance or fluorescence
properties to that of the enzymatic readout. The kinetic assay
reduces the false positive rate. In an additional aspect, the
kinetic rate are normalized to several control wells on each assay
plate. This allows for some variation in the activity of the target
proteins and the stability of assay reagents over time and thus
permits screening runs of several hours.
[0091] When proteins that use ATP are included, the pyruvate
kinase/lactate dehydrogenase embodiments are particularly preferred
due to the advantage of ATP regeneration so that ATP concentration
is constant over time.
[0092] Further regarding variation of the assays, it is understood
that for a kinesin-microtubule modulator assay, the order of
addition of the assay components affects the ATPase rate.
[0093] The invention further provides methods for identifying
whether any two test proteins interact. Briefly, the assay is
functionally similar to a yeast two-hybrid system, but relies on an
increase in ATPase activity as a result of bringing two components
together as a result of a protein-protein interaction. As an
example, the system is described using a biological polymer binding
site and a polymer stimulated ATPase, although as will be
appreciated by those in the art, any two components that result in
an increase in ATPase activity as a result of association can be
used. For example, a first test protein (a "bait" protein), for
which an interaction is sought, is joined, usually covalently, to a
biological polymer binding protein, for example a cytoskeletal
binding protein (such as a microtubule binding protein) to form a
first target chimera. The term "chimera" or "fusion protein" as
used herein refers to a protein (polypeptide) composed of two
polypeptides that, while typically unjoined in their native state,
typically are joined by their respective amino and carboxyl termini
through a peptide linkage to form a single continuous polypeptide.
It will be appreciated that the two polypeptide components can be
directly joined or joined through a peptide linker/spacer.
[0094] A second test protein (a "prey" protein), is joined, again
usually covalently, to an ATPase domain that is stimulated by the
cytoskeletal component to form a second target chimera. Upon
combination with the cytoskeletal component, the first target
chimera binds to the cytoskeletal component, and if the first and
second target proteins interact, the second target chimera is
brought into proximity with the cytoskeletal component, and thus
the ATPase activity is stimulated and can be detected. If there is
no interaction, no increase in ATP production is observed.
[0095] In a preferred embodiment, the biological polymer binding
protein comprises just a domain of a larger protein that comprises
an ATPase domain; that is, the ATPase domain has been removed.
Alternatively, the biological polymer binding protein may include
the larger protein but have the ATPase domain inactivated, for
example by mutation. Similarly, the ATPase domain may be either
just the ATPase functional domain of a protein, or it may include a
larger protein that has the binding domain inactivated.
[0096] As discussed above, the chimera proteins are generally
joined covalently, for example by making fusion proteins, although
covalent cross-linking can be used, or high affinity non-covalent
associations can also be done, for example using binding partners
such as biotin/avidin, etc. In a preferred embodiment, the fusion
proteins are made using fusion genes, as is generally known in the
art.
[0097] In a preferred embodiment, the target proteins should not
have ATPase activity themselves, although it is possible to detect
increases in activity.
[0098] Suitable biological polymers include, but are not limited
to, nucleic acids including DNA and RNA, and cytoskeletal
components including, but not limited to, microtubules and
microfilaments (actin filaments).
[0099] Suitable biological binding sites include, but are not
limited to, nucleic acid binding domains (when nucleic acids are
the biological polymer), and molecular motor binding domains (in
the case of cytoskeletal components).
[0100] Suitable ATPases include, but are not limited to, those that
exhibit an increase (stimulation) in the presence of the
biopolymer, such as DNA and RNA polymerases in the case of nucleic
acids, microtubule stimulated ATPases in the case of microtubules
including kinesins and dyneins, and actin stimulated ATPases such
as myosins.
[0101] In a preferred embodiment, the first test protein is
attached to a functional molecular motor binding domain to provide
a first target chimera. The second test protein is attached to a
functional microtubule stimulated ATPase domain to form a second
target chimera. The first and second target chimeras are combined
under conditions which normally allow activity of a motor protein
which comprises a molecular motor binding domain and a microtubule
stimulated ATPase domain. An increase in motor protein activity
indicates interaction between the two test proteins.
[0102] Customarily one bait protein is used to test a library of
test sequences as is described below; however, as will be
appreciated by those in the art, the bait protein may be one of a
library as well, thus forming an experimental matrix wherein two
libraries (although the coding regions of the libraries could be
identical) are evaluated for protein-protein interactions. In a
preferred embodiment, self-activating bait proteins are filtered
out from the bait protein library.
[0103] In another embodiment a method for identifying whether a
candidate agent is a modulator of at least one of a first and
second test protein is provided. In this case, a candidate agent is
combined with the first and second chimeras as described above. A
change in molecular motor activity in the presence and absence of
the candidate agent indicates that the candidate agent is a
modulator of at least one of the two candidate agents.
[0104] Thus, the chimeras of the present invention can be formed
used recombinant techniques known in the art. The chimera can be
formed at the protein level wherein two polypeptides are joined, or
at the molecular level wherein a nucleic acid is formed which
encodes the appropriate functional motor component and the
appropriate test protein.
[0105] In a preferred embodiment, the nucleic acids encoding a
chimera are used to express the respective recombinant chimera. A
variety of expression vectors, including viral and non-viral
expression vectors can be made which are useful for recombinant
protein expression in a variety of systems, including, but not
limited to, yeast, bacteria, archaebacteria, fungi, insect cells
and animal cells, including mammalian cells.
[0106] The expressed chimera may also include further fusion
domains including tag polypeptides. Recombinant protein is produced
by culturing a host cell transformed with a nucleic acid encoding
the chimera (generally as an expression vector), under the
appropriate conditions that induce or cause expression of the
chimera.
[0107] In a preferred embodiment, the recombinant chimera is
purified following expression, as outlined above.
[0108] For using the chimeras in the assays described herein, if
the two test proteins bind to one another, a complex with both
chimeras comprising a functional molecular motor is formed. Thus,
the binding interaction between the two test proteins can be
identified by functional motor activity under conditions which
would normally allow motor activity if both a functional
microtubule stimulated ATPase and binding domain were present.
[0109] In the case of identifying a modulator in an assay utilizing
the chimeras of the present invention, the modulator can be an
activator of the motor activity. Thus, in the absence of the
candidate agent, there may be no motor activity, however, in the
presence of the candidate agent, motor activity occurs. Conversely,
there may be significant motor activity, indicating that the two
testbinding proteins interact, but this may decrease in the
presence of a candidate agent. In either case, the candidate agent
is identified as a modulator of at least one the two test
proteins.
[0110] In a preferred embodiment, motor activity is identified by
ATP hydrolysis as described above. However, it is understood that
motor activity can be identified by a number of assays. Such assays
include microtubule gliding, depolymerization/polymerization and
any motor activity which requires both binding and ATPase activity.
Therefore, in the case that the molecular motor used has another
specific activity, such as involvement in mitosis or axonal
transport, specific assays for those activities can be
utilized.
[0111] Generally motility assays involve immobilizing one component
of the system (e.g., the kinesin motor or the microtubule) and then
detecting movement, or change thereof, of the other component.
Thus, for example, in a preferred embodiment, the microtubule will
be immobilized (e.g., attached to a solid substrate) and the
movement of the kinesin motor molecule(s) will be visually
detected. Typically the molecule that is to be detected is labeled
(e.g., with a fluorescent label) to facilitate detection.
[0112] Methods of performing motility assays are well known to
those of skill in the art (see, e.g., Hall, et al. (1996), Biophys.
J, 71: 3467-3476, Turner et al., 1996, Anal. Biochem. 242 (1):20-5;
Gittes et al., 1996, Biophys. J. 70(1): 418-29; Shirakawa et al.,
1995, J. Exp. Biol. 198: 1809-15; Winkelmann et al., 1995, Biophys.
J. 68: 2444-53; Winkelmann et al., 1995, Biophys. J. 68: 72S, and
the like).
[0113] In addition to the assays described above for identifying
ATPase activity, conventional methods can be used. For example,
P.sub.i release from kinesin can be quantified. In one preferred
embodiment, the ATPase activity assay utilizes 0.3 M PCA
(perchloric acid) and malachite green reagent (8.27 mM sodium
molybdate II, 0.33 mM malachite green oxalate, and 0.8 mM Triton
X-100). To perform the assay, 10 .mu.L of reaction is quenched in
90 .mu.L of cold 0.3 M PCA. Phosphate standards are used so data
can be converted to mM inorganic phosphate released. When all
reactions and standards have been quenched in PCA, 100 .mu.L of
malachite green reagent is added to the to relevant wells in e.g.,
a microtiter plate. The mixture is developed for 10-15 minutes and
the plate is read at an absorbance of 650 nm. If phosphate
standards were used, absorbance readings can be converted to mM
P.sub.i and plotted over time.
[0114] Additionally, in the case of methods provided herein
utilizing the chimeras in accordance with the present invention,
the remaining ATP can be measured using the luciferin-luciferase
system. Anal. Biochem., 40:1-17 (1971).
[0115] The assays are preferably performed in a high throughput
system as described herein utilizing multiwell plates and
fluorescence or absorbance readouts.
[0116] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety.
EXAMPLE
A High Throughput Assay for Modulators of the Molecular Motor
Kinesin
[0117] This assay is based on detection of ADP production from
kinesin's microtubule stimulated ATPase. ADP production is
monitored by a coupled enzyme system consisting of pyruvate kinase
and lactate dehydrogenase. Under the assay conditions described
below, pyruvate kinase catalyzes the conversion of ADP and
phosphoenol pyruvate to pyruvate and ATP. Lactate dehydrogenase
then catalyzes the oxidation-reduction reaction of pyruvate and
NADH to lactate and NAD+. Thus, for each molecule of ADP produced,
one molecule of NADH is consumed. The amount of NADH in the assay
solution is monitored by measuring 6 light absorbance at a
wavelength of 340 nm.
Assay Components
[0118] A kinesin heavy chain construct consisting of the N-terminal
420 amino acids is used in the assay. The final 25 .mu.l assay
solution consists of the following: 5 .mu.g/ml kinesin, 30 .mu.g/ml
microrubules, 5 .mu.M Taxol, 0.8 mM NADH, 1.5 mM phosphoenol
pyruvate, 3.5 U/ml pyruvate kinase, 5 U/ml lactate dehydrogenase,
25 mM Pipes/KOH pH 6.8, 2 mM MgCl.sub.2, 1 mM EGTA, 1 m MDTT, 0.1
mg/ml BSA, 0.001% antifoam 289 (Sigma), and 1 mM ATP.
Compound Plates
[0119] Potential chemical modulators of kinesin are dissolved in
DMSO at a concentration of approximately 1 mg/ml, and 0.5 .mu.l of
each chemical solution is dispensed into a single well of a clear
384 well plate (Clinipate, Labsystems). On each plate, there are at
least 16 wells into which pure DMSO (without a candidate compound)
is dispensed. These wells serve as negative controls for comparison
to the potential chemical modulators on that plate. The compound
plates are made in advance and stored at 4.degree. C., and each
plate is labeled with a bar code which is used to identify the
compounds on a given plate.
Instrumentation
[0120] The robotic system that runs the assay consists of a plate
storage and retrieval device (Plate Stak, CCS Packard), a 96
channel automated pipetting device (Multimek, Beckman), a robotic
arm (Twister, Zymark), and a plate reader for absorbance
(Ultramark, BioRad). The system is controlled by a custom-built
software application.
Assay Performance
[0121] A stack of compound plates is placed in the plate storage
devices and plates are transferred one at a time to the automated
pipetting device by the plate carrier of the Plat Stak. Each of the
384 wells are then filled with 20 .mu.l of a solution consisting of
all of the assay components described above except for ATP. The
plate is then agitated at high frequency by rapidly moving the
plate carrier between two positions that are separated by a few
millimeters. The plate is then returned to the pipetting position.
While the shaking of the plate occurs, the pipet tips are washed
with a solution of 0.001% antifoam in deionized 6 water. To start
the assay, 5 .mu.l of a second solution containing ATP is then
added to each well. The solution is then mixed by a second cycle of
high frequency agitation. The plate is then transferred to the
plate reader by the robotic arm. In the plate reader, 10 absorbance
measurements at 340 nm are taken at 12 second intervals to produce
a 2 minute kinetic read for each well. While one plate is being
read, the next plate is transferred to the pipetting device and
prepared up to but not including the addition of the second
solution. When the plate read is complete, the robotic arm
transfers the plate to a waste chute and simultaneously the second
solution is pipetted into the next plate so that it can be
transferred to the reader to complete the cycle. The entire assay
is run at room temperature 20.degree. C.
Data Analysis
[0122] Following data acquisition, the maximum rate of the
absorbance change is calculated for each well and normalized to the
average of the control wells (without compound) which were present
on the same plate. The normalized rates are then entered into an
Oracle database, and this allows them to be correlated with the
potential chemical modulators. On 21 each plate, the coefficient of
variation of the slopes for the control wells ranges from 4-8%.
Quality control is assured by monitoring for a minimal initial
absorbance and a linear absorbance change.
Important Features
[0123] There are several features of this system which are
important. The kinetic design which consists of multiple absorbance
measurements dramatically improves the specificity of the assay
over a single endpoint measurement. First, the rate of the reaction
is to a first approximation independent of small differences
between wells in the time from the start of the reaction to the
first reading, and as a result, the overall variation in the data
is reduced. Second, the rate of the absorbance change is not
affected by having a chemical compound which absorbs light of the
same wavelength.
[0124] The presence of control wells in each plate and the
subsequent normalization of the data to those wells allows data to
be taken for several hours despite some degradation of the enzyme
activities which results from the aging of the solutions. This also
improves the reproducibility of the data.
[0125] The presence of antifoam in the solution and the tip washing
solution improves overall liquid handling by reducing the number of
trapped bubbles in the small wells and helps flatten the fluid
meniscus in each well for more reliable absorbance measurements.
Additional features which improve liquid handling are the vigorous
shaking of the plate described above, and the round shape of the
wells in the microplates used.
[0126] The assay components and the performance of the assay are
optimized together to match the overall read time with the rate of
kinesin's ADP production. In this example, the rate of absorbance
change is approximately 150-250 mOD/min. This corresponds to the
production of approximately 2 .mu.M ADP/sec. In addition to
optimizing the rate of ADP production, the read time must be long
enough for the rate of NADH consumption to reach steady state
beyond an initial lag time of several seconds. In some cases, the
order of addition of the reagents can have a significant affect on
the rate of ADP production. In the above example, the optimal rate
is achieved by premixing all reagents except for the compound of
interest and ATP.
* * * * *